Abstract: Aspergillus ochraceus contamination was detected in the forty six rice samples collected from areas exposed to rain or flood or stored in godown or mills. These isolates were grown on Pusa basmati rice grains for 7 days at room temperature than extracted the Ochratoxin A (OTA) and identified on TLC plate along with standard in different solvent systems. The OTA was confirmed by changing the colour from greenish blue to blue after spraying 6% NaOH in 20% ethanol on TLC plate. Garlic bulb extract (10%) proved significantly more effective with complete inhibition of A. ochraceus. Pongamia glaberima kernel extract (20%) showed 71% inhibition on mycelial growth of A. ochraceus. Among the culture filtrate of the biocontrol agent, Trichoderma virens inhibited completely while the culture filtrate from T. harzianum showed a partial inhibition (67%) at 15% concentration. The culture filtrate of Pseudomonas fluorescens (DRPf 002) were effectively inhibited (98%) mycelial growth at 15% concentration. Among the fungicides tested carbendazim was effective even at 100 ppm in inhibiting A. ochraceus. Propineb completely reduced the growth at 750 ppm. Among the chemicals tested benzoic acid were completely inhibited the mycelial growth at 0.1% concentration.
INTRODUCTION
Rice (Oryzae sativa L.) is the most important staple food crop in India and bulk of rice is grown in kharif or wet season. Frequent and heavy rainfall and floods particularly near harvest in coastal areas in eastern, southern and western regions of the country wet the crop and make panicles more prone to invasion by fungi and bacteria. As the harvested sheaves are left in moist fields over strubbles to dry, the grains become infected by Aspergillus sp. (Reddy et al., 2004). The mycotoxin producing fungi viz., Aspergillus sp., Penicillium sp. and Fusarium sp. were found in the seed samples rough rice stored in government warehouses (Carlos et al., 2000). Species of Aspergillus, Penicillium, Fusarium and Alternaria were the most important mycotoxigenic fungi in rice grains (Shetty et al., 1994). Indeed, among more than 100,000 fungal species able to contaminate feeds and foods, some have the ability to synthesize molecules toxic for human and animals. Rice represents a very good substrate for fungal growth and toxinogenesis since it is used as an ideal culture medium to test the toxigenic potential of isolated strains (Bars and Bars, 1992). Some surveys showed that this basic food might be contaminated by mycotoxins such as Aflatoxins and Ochratoxin A (Begum and Samajpati, 2000). The Ochratoxin A contamination was identified in rice purchased from retail shops in the Rabat and Sale area in Morocco (Zinedine et al., 2007). That contamination may represent a real threat to human health specially concerning chronic toxicity of these toxins, which are known or suspected to be carcinogenic. However, detailed information on OTA contamination in rice is not available. We therefore made an attempt to isolate OTA producing mycoflora in the samples collected from different rice growing areas of India and evaluate plant extracts, biocontrol agents, fungicides and chemicals to limit the growth of A. ochraceus.
MATERIALS AND METHODS
This study was carried out from February 2005 to October 2006 at Plant Pathology Laboratory, Directorate of Rice Research, Rajendranagar, Hyderabad, A. P, India. Five hundred and five seed samples were collected from 36 locations in 21 states in the country (Andaman and Nicobar islands, Andhra Pradesh, Assam, Bihar, Chattishgarh, Gujarat, Himachal Pradesh, Jammu and Kashmir, Karnataka, Kerala, Madhya Pradesh, Maharashtra, Meghalaya, Orissa, Pondicherry, Punjab, Rajasthan, Tamil Nadu, Tripura, Uttaranchal and West Bengal). Using agar plate method OTA producing mycoflora was isolated from these samples. Seeds were surface sterilized with chlorax (0.1%) and washed twice with distilled water. Two hundred seeds were plated on one-half strength potato dextrose agar medium having Rose Bengal (50 ppm) (Cotty, 1994). The plates were incubated at room temperature and presence of A. ochraceus was observed after six days.
For identification of OTA, A. ochraceus isolates were grown on pusa basmati rice grains were soaked 2 h in tap water and fermentation was carried out in 500 mL Erlenmeyer flasks containing 50 g of rice inoculated with 1 mL of spore suspension of A. ochraceus was originally isolated from rice grains collected from different rice growing areas of India. Three treatments were maintained for each strain. After 7 days of fungal growth on rice grains, than extracted the OTA through the moldy rice (25 g) was blended in waring blender for 5 min with Acetonitrile-4% KCl-6N HCl (88: 10:2) (Eucaris et al., 1982). The mixture was filtered through Whatman No. 1 filter paper and concentrated on a rotary evaporator to 1 mL. OTA production was evaluated by standard thin layer silica gel chromatography to confirm the presence or absence of OTA along with standard. The standard was procured from sigma chemicals. Two replicates were analyzed by spotting crude extract of OTA. The TLC plates used were coated with silica gel 60 F254 on aluminum sheet, 20x20 cm and the silica gel plates were developed in the following solventsystems (vol/vol): (A) toluene-ethyl acetate-formic acid (6:3:1), (B) benzene-methanol-acetic acid (90:5:5), (C) chloroform-ethyl acetate-formic acid (10:3:1), (D) chloroform-acetone-formic acid (6:2:1), (E) benzene-ethyl acetate-formic acid (3:1:1), (F) benzene-aceticacid (4:1) and (G) chloroform-diethyl ether-acetic acid (17:5:1). The plates were than observed under UV light at 365nm and calculated Rf value. The OTA was confirmed on TLC by changing the colour from greenish blue to blue after spraying 6% NaOH in 20% ethanol (Golinski and Grabarkiew, 1984).
Plant extracts viz., Allium sativum bulb extract, Annona squamosa kernel extract, Allium cepa bulb extract, Pongamia glabarema kernel extract, Azadirachta indica leaf and kernel extract and Eucalyptus terticornis leaf extract, biocontrol agents viz., Trichoderma viride (MTCC 800), T. harzianum (MTCC 2050), T. virens (MTCC 794), T. reseii (MTCC 2480) and T. koningi (MTCC 796), five strains of Pseudomonas fluorescens, fungicides viz., propineb 70 WP, bitertanol 25 WP, carbendazim 50 WP and tricyclazole 75 WP and chemicals viz., acetic acid, benzoic acid, propionic acid, vanillin and sodium chloride were tested for their efficacy on the growth of A. ochraceus. The Trichoderma cultures were grown in potato dextrose broth for seven days at 28°C and the cultures were filtered through Whatman No. 1 filter paper to get cell-free filtrate. The Pseudomonas cultures were grown in King’S B broth for 4 days in 28°C at 120 rpm and the cultures were centrifuged at 10,000 rpm for 10 min to get cell-free filtrate.
Similarly clear extracts from plants (Pongamia, onion, custard apple, Neem kernel and garlic @ 25 g/100 mL and Eucalyptus and neem leaf @ 3 g/100 mL water) were also obtained. The extracts of each plant species at 5, 10, 15 and 20% and biocontrol agents at 5, 10 and 15% were tested by poison food technique (Nene and Thapliyal, 1971). Fungicides were tested at 100, 250, 500, 750 and 1000 ppm concentration and chemicals at 0.1, 0.2, 0.3, 0.4% concentration. The medium incorporated with desired treatment was poured into petri dishes, allowed to solidify and a loop full of inoculum of OTA producing culture was transferred. PDA without plant extract, fungicide or chemical served as control and three replications were maintained for each treatment.
RESULTS
The level of infection of A. ochraceus varied from 1 to 100% in the
46 rice samples collected from different rice growing regions of India (Table 1). Forty-six isolates of A. ochraceus were obtained from these samples.
On an average, the infection with A. ochraceus was to the extent of 65.3%
in the samples collected from Pusa where the seed was stored for one to two
years in the godowns and open exposed to flood. A similar level of infection
was found in the seed samples that were exposed to rain at Arundhatinagar (Tripura).
However, the seed stored for one to two years in godowns showed 46.5% at Titabar
(Assam). The seed stored for 2 years in godowns showed 38.5% at Megalaya (Gujarat).
The occurrence of A. ochraceus in the seed samples open exposed to rain
was 35.5% at arundathinagar (Tripura) and 28.5% at Aduthurai (Tamil Nadu). The
other samples showed low level of infection from 1.4 to 11% were the seeds stored
in godowns, damp condition and the seed collected from standing crop. Fifteen
isolates of A. ochraceus were purified by single spore isolation and
named as DRAo 001 to DRAo 015. Ochratoxin A were isolated from the A. ochraceus
isolates (DRAo 008 and DRAo 013) and identified on TLC plates by using the different
solvent systems. The OTA isolated in this study and standard showed the same
Rf values (Table 2) in different solvent systems. The OTA was confirmed
on TLC plate by changing the colour from greenish blue to blue after spraying
the 6% NaOH in 20% ethanol.
| Table 1: | Occurrence of Aspergillus ochraceus on seed samples |
| Table 2: | Rf value of ochratoxin A in different solvent systems |
Garlic bulb extract (10%) completely inhibited the fungal growth of the A. ochraceus while the efficacy of other plant products was only in the range of 61 to 71% even at 20% concentration (Table 3). Pongamia kernel extract gave good control (71%) at 20% concentration. But, neem kernel extract (0-69%), neem leaf extract (0-66%), custard apple kernel extract (0-64%) Eucalyptus and onion leaf extract (7-61%) were less effective in inhibiting the growth of A. ochraceus.
Of the Trichoderma isolates evaluated against A. ochraceus, culture
filtrate of T. virens showed a complete inhibition of growth at
15% concentration and was significantly superior over all other Trichoderma
sp. (Table 4). The order of performance was T. harzianum, which showed
a moderate inhibition (37-67%) followed by T. koningi (8-38%), T.
viride (10-38%), T. reseii (9-32%) and among the five strains of
Pseudomonas fluorescens culture filtrates the DRPf 002 were effectively
inhibited the growth (98%) at 15% concentration followed by DRPf 005 (93%).
The others are less effective in reducing the growth of A. ochraceus even
at 15% concentration (28-51%) (Table 5).
| Table 3: | Effect of plant extracts on the mycelial growth of A. ochraceus |
| RG-Radial growth (mm), INH- % Inhibition over control | |
| Table 4: | Effect of Trichoderma culture filtrates on mycelial dry weight of A. ochraceus |
| MDW- Mycelial dry weight (g), INH-% Inhibition over control | |
| Table 5: | Effect of Pseudomonas fluorescens culture filtrates on mycelial weight of A.ochraceus |
| MDW- Mycelial dry weight (g), INH-% Inhibition over control | |
| Table 6: | Effect of fungicides on mycelial growth of A. ochraceus |
| RG- Radial growth (mm), INH-% Inhibition over control | |
All the fungicides were significantly effective in reducing the growth of A.
ochraceus over control (Table 6). Of the test fungicides, carbendazim was
significantly effective (100%) even at 100 ppm.
| Table 7: | Effect of Chemicals on mycelial growth of A. ochraceus |
| RG- Radial growth (mm), INH- % Inhibition over control | |
Among the other test formulations, propineb was found highly effective at 750 ppm, where it had shown 100% inhibition of the test Aspergilli. However, the efficacy of tricyclazole and bitertinol was only in the range of 53 to 100% on A. ochraceus at the highest concentration tested. The fungicidal effect was more pronounced in all the test fungicides with any increase in concentration from 100 to 1000 ppm. Among the chemicals benzoic acid were completely inhibited the mycelial growth at 0.1% followed by acetic acid at 0.2% concentration (Table 7).
DISCUSSION
The data on survey revealed A. ochraceus in 46 rice samples. The infection of A. ochraceus dominated in the samples collected from Pusa. These results confirm the earlier observations of Trung et al. (2001) had also reported the A. ochraceus as one of the most predominant fungi in the rice samples collected from mills of Mekong delta. Canton, Vietman. Reddy (1990) had also reported Aspergillus sp. as one of the most predominant fungus in the grain samples of flood affected paddy variety NLR 9672 collected from standing crop, threshing floors and storage sites in Nellore district of Andhra Pradesh.
Eucaris et al. (1982) used a TLC based technique for the estimation and quantification of Ochratoxin A in rice and other vegetable foods. Golinski and Grabarkiew (1984) developed TLC based technique for the confirmation of Ochratoxin A on TLC by spraying of NaOH. Santosh and Vargas (2002), Fredrik et al. (1981) also developed TLC for detection of OTA by using different solvent systems. We also used the Eucaris method for extraction and Fredrik method for identification of OTA produced by Aspergillus ochraceus isolates.
Arun et al. (1995) reported the fungitoxic effect of garlic against Aspergillus niger and Fusarium pallidoroseum in pigeon pea. Our study on effect of plant extracts on A. ochraceus showed that garlic bulb at 10% concentration was the most effective treatment in reducing the growth compared to other plant products. The fungitoxicity of the garlic bulb extract in this study might be due to antifungal substances present in garlic namely sulphur containing compounds like allicin, allylpropyl disulphide or diallyl disulphide, or due to an enzyme allinase (Radha et al., 1998).
The studies on antagonistic effect of culture filtrates of five Trichoderma species against A. ochraceus showed that at 15% concentration, Trichoderma virens was the most effective in reducing the growth of test Aspergilli compared to other culture filtrates. Calistru et al. (1997) has also reported a similar potential for biological control of A. ochraceus using Trichoderma sp., based on in vitro results.
Shekhawat et al. (1986) had reported the effective control of the seed borne inoculum in groundnut following the seed treatment with a higher dose of carbendazim (2.5 g kg-1). This study however, showed that carbendazim even at 100 ppm concentration was the most effective fungicide in reducing the growth of A. ochraceus compared to the other fungicides. Chipley and Uraih (1980) had reported the control of Aspergillus sp. and Aflatoxin release by derivarives of benzoic acid. In this study we found that the benzoic acid were completely inhibited the mycelial growth of A. ochraceus at 0.1% concentration.
Present study had shown that plant extract like garlic bulb, biological agents like Trichoderma virens and Pseudomonas fluorescens, fungicide like carbendazim and chemical like benzoic acid prevent or drastically reduce the incidence of A. ochraceus on rice/grains during storage. Therefore, further studies are warranted to deploy them usefully as seed/grain treatment to avoid the occurrence of OTA contamination in grains and chemical characterization of OTA and development of antibodies and ELISA technique to check the OTA contamination by qualitatively and quantitatively in rice grains.
ACKNOWLEDGMENTS
The authors are thankful to Indian Council of Agricultural Research, New Delhi for providing facilities through a network project. We also thank Microbial Type Culture Collection, IMTECH Chandigarh for providing cultures of Trichoderma sp.